CN107849088A - The purification process of antibody-like protein matter - Google Patents
The purification process of antibody-like protein matter Download PDFInfo
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- CN107849088A CN107849088A CN201680042481.XA CN201680042481A CN107849088A CN 107849088 A CN107849088 A CN 107849088A CN 201680042481 A CN201680042481 A CN 201680042481A CN 107849088 A CN107849088 A CN 107849088A
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- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
- B01D15/3804—Affinity chromatography
- B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G, L chromatography
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
- B01J20/3206—Organic carriers, supports or substrates
- B01J20/3208—Polymeric carriers, supports or substrates
- B01J20/3212—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
- B01J20/3217—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
- B01J20/3219—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond involving a particular spacer or linking group, e.g. for attaching an active group
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- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
- B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
- B01J20/3268—Macromolecular compounds
- B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
- B01J20/3274—Proteins, nucleic acids, polysaccharides, antibodies or antigens
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
- C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/16—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the fluid carrier
- B01D15/166—Fluid composition conditioning, e.g. gradient
- B01D15/168—Fluid composition conditioning, e.g. gradient pH gradient, chromatofocusing, i.e. separation according to the isoelectric point pI
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
- C07K17/02—Peptides being immobilised on, or in, an organic carrier
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Abstract
The present invention provides the purification process of the antibody-like protein matter comprising the elution procedure under solutions of weak acidity.The present invention provides a kind of purification process of antibody-like protein matter, and it includes following (a)~(b) process:(a) antibody-like protein matter is made to be contacted with the affine isolation medium containing the part for being immobilized onto carrier, so that the process that antibody-like protein matter is adsorbed in affine isolation medium;PH3.5 more than eluent with affine isolation medium is contacted, process that antibody-like protein matter eluted (b);Aforementioned ligand contains the Gln of Fc binding sites and/or Lys be replaced into Ala, Ser and/or Thr in E, D, A, B or the amino acid sequence in C-structure domain described in sequence number 1~5 from albumin A obtained from amino acid sequence, and the part, compared with the part before foregoing displacement, the antibody binding capacity in acid pH range reduces.
Description
Technical field
The present invention relates to a kind of purification process for the antibody-like protein matter for including the elution procedure under solutions of weak acidity.
Background technology
The core for carrying out antibody drug exploitation is monoclonal antibody, and these monoclonal antibodies are exactly using restructuring culture cell
What technology etc. was produced in large quantities." monoclonal antibody " refers to anti-by being obtained from the celliferous clone of monospecific antibody production
Body.Carrying out turning into medicine after purification using various chromatograms by the monoclonal antibody that culture cell is produced.In chromatogram, particularly
The purifying that the affine separation chromatography for having albumin A using immobilization is carried out disposably can purify antibody from animal cell culture
For high-purity, therefore it is essential process in the manufacture of antibody medicine.
Albumin A is produced by the staphylococcus aureus (Staphylococcus aureus) in gram-positive bacteria
1 kind of cell wall protein, by signal sequence S, (E domains, D domains, A are tied 5 immunoglobulin associativity domains
Structure domain, B structure domain, C-structure domain) and as cell wall binding domain XM areas form (non-patent literature 1).
A variety of transformed albumin A by protein engineering mode, so that its multifunction is developed
Technology.Such as the known alkali resistance for improving albumin A, improve antibody acid dissociation characteristic, by importing variation to immobilization point to carry
The examples such as high antibody binding capacity (patent document 1~4).
In recent years, the production potency of the antibody in cell culture is being improved, and the burden of the purification process in downstream is also being increased
Add.In the process for the affine separation chromatography for having albumin A using immobilization, generally make antibody binding under neutral ph in carrier
Afterwards, antibody elution at acidic, so as to by antibody purification.However, it is known that there is partial antibody to gather at a low ph
Collection or antibody activity reduce.The burden that these phenomenons not only turn into purification procedures in antibody manufacture (process number increase, is received
Rate reduces), and bring serious side effect sometimes as medicine.It is therefore desirable to be able to what is eluted under higher pH is affine
Separation chromatography carrier.The known Ser for carrying out 33 to improve antibody acid dissociation characteristic displacement, the His of 18 displacement
And various radical amino acid replacements are the schemes (patent document 3,5,6) such as His.
Although corresponding to the C-structure domain in the Fc binding sites of albumin A the Gln of the 9th to Ala, Thr replacement mutation
It is known, but does not disclose the antibody acid dissociation characteristic (patent document 7, non-patent literature 2) of these variants.It is in addition, right
Should have known to replacement mutation body in the Lys of the 35th of the C-structure domain in the Fc binding sites of albumin A a variety of, but these become
The antibody acid dissociation characteristic of allosome does not disclose (patent document 8).
Prior art literature
Patent document
Patent document 1:International Publication No. 03/080655
Patent document 2:No. 1123389 specifications of European Patent No.
Patent document 3:International Publication No. 2011/118699
Patent document 4:International Publication No. 2012/133349
Patent document 5:International Publication No. 2012/087231
Patent document 6:International Publication No. 2012/165544
Patent document 7:International Publication No. 2015/005859
Patent document 8:Japanese Unexamined Patent Publication 2007-252368 publications
Non-patent literature
Non-patent literature 1:The work such as Hober S., " J.Chromatogr.B " 2007, volume 848,40-47 pages
Non-patent literature 2:O ' Seaghdha M. etc. write, " FEBS J ", 2006, volume 273,4831-4841 pages
The content of the invention
Problems to be solved by the invention
Problem of the invention is that, there is provided the purifying side of the antibody-like protein matter comprising the elution procedure under solutions of weak acidity
Method.
The solution used to solve the problem
The activity of various recombinant protein A variant of the present inventors to being made a variation with amino acid replacement compares
Research, as a result finds, containing in E, D, A, B or the amino acid sequence in C-structure domain described in sequence number 1~5 from albumin A
The part of amino acid sequence obtained from the Gln of Fc binding sites and/or Lys are replaced into Ala, Ser and/or Thr, in acidity
Antibody binding capacity in the range of pH reduces compared with the part before foregoing displacement, so as to complete the present invention.
That is, the present invention relates to a kind of purification process of antibody-like protein matter, it includes following (a)~(b) process:(a)
Antibody-like protein matter is set to be contacted with the affine isolation medium containing the part for being immobilized onto carrier, so that antibody-like protein matter is inhaled
The process for investing affine isolation medium;PH3.5 more than eluent with affine isolation medium is contacted, by antibody sample egg (b)
The process of white matter elution;Aforementioned ligand contains in E, D, A, B or the ammonia in C-structure domain described in sequence number 1~5 from albumin A
Amino acid sequence obtained from the Gln of Fc binding sites and/or Lys are replaced into Ala, Ser and/or Thr in base acid sequence, institute
Part is stated compared with the part before foregoing displacement, the antibody binding capacity in acid pH range reduces.] preferably foregoing affine point
It is the carrier for having in water-insoluble base material immobilization part from matrix.
It is preferred that water-insoluble base material contains synthesis macromolecule or polysaccharide.
Preferred polysaccharide class is cellulose or agarose.
It is preferred that eluent is containing selected from by acetate ion, citrate ion, glycine, succinate ion, phosphorus
The acidic buffer of at least one kind of anionic species in the group of acid ion and formate ion composition.
It is preferred that the aggregation of the antibody-like protein matter of protein from host and/or immunoglobulin in eluent
Content is reduced.
The elution of preferred antibody sample protein is eluted using pH gradient and carried out.
It is preferred that pH gradient elution is carried out using the eluent of pH4~6.
Antibody-like protein matter before purification can be the mixture with the protein from host cell.
Antibody-like protein matter before purification can be the mixture with the aggregation of antibody-like protein matter.
The effect of invention
The present invention antibody-like protein matter purification process can under pH high than ever antibody elution sample protein.
Brief description of the drawings
Fig. 1 is E, D, A, B of the albumin A of staphylococcus (Staphylococcus) and the sequence comparison table in C-structure domain.
Embodiment
The present invention is a kind of purification process of antibody-like protein matter, and it includes following (a)~(b) process:(a) antibody is made
Sample protein contacts with the affine isolation medium containing the part for being immobilized onto carrier, so that antibody-like protein matter is adsorbed in parent
With isolation medium process;PH3.5 more than eluent with affine isolation medium is contacted, antibody-like protein matter eluted (b)
Process;Aforementioned ligand contains in E, D, A, B or the amino acid sequence in C-structure domain described in sequence number 1~5 from albumin A
It is middle the Gln of Fc binding sites and/or Lys are replaced into Ala, Ser and/or Thr obtained from amino acid sequence, the part with
Part before foregoing displacement is compared, and the antibody binding capacity in acid pH range reduces.
Albumin A is included as E, D, A, B of immunoglobulin associativity domain and the protein in C-structure domain.E、D、
A, B and C-structure domain are that the immunoglobulin that can be incorporated into the region beyond the complementary determining region (CDR) of immunoglobulin combines
Property domain, any structure domain is respectively provided with the especially Fv areas in the Fc areas for being incorporated into immunoglobulin, Fab areas and Fab areas
Activity.It should be noted that the source of albumin A is not particularly limited in the present invention, staphylococcus is preferred from
(Staphylococcus) albumin A.
" protein " this term includes all molecules with polypeptide structure, fragmentation or connected by peptide bond
Polypeptide chain is also included within " protein " this term.In addition, " domain " is the unit in the higher structure of protein, refer to
Formed to hundreds of amino acid residue sequences by tens of and be enough to show certain physical chemistry function or biochemical function
The unit of protein.
Amino acid sequence from domain refers to the amino acid sequence before replacement amino acid.Amino acid from domain
Sequence is not limited only to E, D, A, B of albumin A or the wild-type amino acid sequence in C-structure domain, by the replacing of amino acid, inserts
Enter, lack and chemical modification and the partial amino acid sequence transformed and with the binding ability protein with Fc areas, then
Included in the amino acid sequence of domain.As the amino acid sequence from domain, can enumerate for example:Form sequence
E, D, A, B of the albumin A of staphylococcus described in row number 1~5 and the amino acid sequence in C-structure domain, can be enumerated in addition:It is right
The ammonia for the variation that the Gly corresponding to the 29 of C-structure domain is replaced into Ala is imported with E, D, A, B of albumin A and C-structure domain
The protein that base acid sequence is formed.In addition the Z domains to make a variation as A1V and G29A are imported with B structure domain also to have
The binding ability in You Yu Fc areas, therefore fall within the amino acid sequence from domain.Amino acid sequence from domain is excellent
Select the domain or its variant that chemical stability is high.
Amino acid sequence from domain has the binding ability with Fc areas.Amino acid sequence and sequence from domain
E, D, A, B of albumin A described in row number 1~5 or the sequence homology in C-structure domain are preferably more than 85%, and more preferably 90%
More than, more preferably more than 95%.
The part that the present invention uses contains in E, D, A, B or the ammonia in C-structure domain described in sequence number 1~5 from albumin A
Amino acid sequence obtained from the Gln of Fc binding sites and/or Lys are replaced into Ala, Ser and/or Thr in base acid sequence.
In E, D, A, B or the amino acid sequence in C-structure domain described in sequence number 1~5 from albumin A, Fc joint portions
Position refer to the 5th of C-structure domain corresponding to albumin A, the 9th, the 10th, the 11st, the 13rd, the 14th, the 17th, the
28, the 31st, the 32nd, the amino acid residue of the 35th (Proc.Natl.Acad.Sci.Usa, 2000, volume 97,
5399-5404 pages).
As the Gln of Fc binding sites, the 9th corresponding to C-structure domain, the 10th, the amino of the 32nd can be enumerated
Sour residue.Wherein, the 9th, the amino acid residue of the 32nd in C-structure domain are preferably corresponded to.
As the Lys of Fc binding sites, the amino acid residue of the 35th corresponding to C-structure domain can be enumerated.
The displacement of amino acid refers to:Remove original acid and the change of other amino acid of variety classes is added on its position
It is different.It should be noted that the statement of the variation on replacement amino acid, is expressed as charging to wild type before the sequence number of displacement position
Or unmanifest type amino acid, the amino acid to morph is charged to after the sequence number of displacement position.For example, by the Gly of the 29th
The variation for being replaced into Ala is expressed as G29A.
As the amino acid for entering line replacement at the Gln and/or Lys of Fc binding sites, Ala, Ser, Thr can be enumerated.
As more specifically substitute mode, can enumerate:Corresponding to C-structure domain the Gln of the 9th to the replacing of Ala, right
Displacements of the Gln that Ying Yu is 9 to Ser, corresponding to displacements of the Gln of the 9th to Thr, corresponding to the Gln of the 32nd to Ala
Or Thr displacement, corresponding to displacements of the Lys of the 35th to Ser.Wherein, Q9A, Q9S preferably in C-structure domain, Q9T,
Q32A、Q32T、K35S。
On the number of amino acid replacement, as long as the antibody binding capacity compared with before replacing in acid pH range reduces
It is not particularly limited, from the viewpoint of the stereochemical structure of the protein before maintaining variation to import, preferably less than 4, more
Preferably less than 2.
As long as the antibody binding capacity compared with before replacing in acid pH range reduces, then except Fc binding sites
Gln and/or Lys can also contain arbitrary amino acid replacement to beyond Ala, Ser and/or Thr displacement.As such
Amino acid replacement, the G29A, F5A that can be enumerated in C-structure domain, F5Y, A12R, F13Y, L17I, L17T, L17V, L19R,
L22R, Q26R, I31L, I31S, I31T, I31V, Q32R, S33H, V40Q, V40T, V40H displacement.In addition can enumerate E, D,
Displacement in A or B structure domain, same with the amino acid of the aforementioned location corresponding to C-structure domain.In addition, Asn is replaced into it
The amino acid replacement of its amino acid can expect the raising of alkali resistance, therefore be preferable.
By Fc joint portions in E, D, A, B or the amino acid sequence in C-structure domain described in sequence number 1~5 from albumin A
Amino acid sequence obtained from the Gln and/or Lys of position are replaced into Ala, Ser and/or Thr and the albumin A described in sequence number 1~5
E, D, A, B or the sequence homology in C-structure domain be preferably more than 85%, more preferably more than 90%, more preferably
More than 95%.
In the part that the present invention uses, more than 90% in amino acid residue preferably as shown below is retained, more excellent
More than 95% is selected to be retained:Gln-9、Gln-10、Phe-13、Tyr-14、Leu-17、Pro-20、Asn-21、Leu-22、Gln-
26th, Arg-27, Phe-30, Ile-31, Leu-34, Pro-38, Ser-39, Leu-45, Leu-51, Asn-52, Gln-55 and
Pro-57 (residue number corresponds to C-structure domain).
The part that the present invention uses, it is characterised in that compared with before displacement, the antibody binding capacity in acid pH range
Reduce.As acid pH range, the scope of faintly acid scope, specially pH3~6 can be enumerated.
Antibody binding capacity in acid range can be by using the pH gradient elution test (embodiment of IgG agaroses
1), antibody binding capacity is determined using intermolecular interaction resolver to evaluate in acid pH range.For example, using
In the case of the pH gradient elution test of IgG agaroses, compared with the protein of (such as C-G29A.2d) before variation imports, acid
Property in the range of antibody binding capacity reduce variant can be eluted under higher pH.With by the egg before variation importing
When on the basis of the elution pH that the summit of the eluting peak of white matter calculates, the elution pH of variant is preferably high by more than 0.05, more preferably high
More than 0.1.
The part that the present invention uses can be the part for only including the single structure domain for being imported with aforementioned amino acid displacement,
It can be matching somebody with somebody comprising multiple domains obtained from the domain for being imported with aforementioned amino acid displacement of more than 2 is connected
Body.
When for part comprising multiple domains when, part both can be part (the equal dimerization being made up of homogenous configuration domain
The homopolymers such as body, equal tripolymer) or part (heterodimer, heterotrimer etc. for being made up of different types of domain
Heteromer).The number of the domain connected is preferably more than 2, more preferably 2~10, more preferably 2~
6.
In the part comprising multiple domains, as the mutual connected mode of monomer domain, it can enumerate:Not by
The method being directly connected to as the amino acid residue of joint;Or the method connected by one or more amino acid residues, but not
It is limited to these.The number of amino acid residue used in connection is not particularly limited, as long as connected mode and connection number are not
It can cause the 3-dimensional stereochemical structure of monomer domain is unstable to be not particularly limited.
In addition, the fusion egg formed using aforementioned ligand as one of constituent, the other oroteins different from function fusion
It can also use in the present invention in vain.As the example of fusion protein, can enumerate fusion have albumin, GST (glutathione S-
Transferase), MBP (maltose-binding protein) protein as an example, but be not limited to these.By to melt with GST, MBP
Hop protein form is expressed, so as to easily carry out the purifying of part.Fitted in addition, DNA can also have been merged in part
The macromolecules such as the nucleic acid such as body, antibiotic etc, PEG (polyethylene glycol).
On encoding the DNA of above-mentioned part, as long as amino acid sequence obtained by the base sequence translation for forming it is formed
Part.Such base sequence can utilize usually used known method, such as polymerase chain reaction (hereinafter referred to as
PCR) method obtains.Additionally it is possible to be synthesized by known chemical synthesis, and then can also be obtained by DNA library.The alkali
The codon of basic sequence can be degenerated into codon substitutions, as long as coding then need not be with original base sequence with monoamino-acid during translation
Arrange identical.
The DNA of the present invention passes through the known DNA sites to the domain of encoding wild type or the albumin A of anomaly
Specifically import variation and obtain.The importing of site-specific mutation can use recombinant DNA technology, PCR in such a way
Method etc. is carried out.
The variation carried out using recombinant DNA technology is imported and can for example carried out by boxlike alternative method, and the boxlike becomes
Different method refers to:It is desirable that the both sides for importing the target site of variation have appropriate restriction enzyme identification in the gene of coding part
During sequence, these limitation enzyme recognition sequence parts are cut off with foregoing restriction enzyme and removed comprising the site for it is expected importing variation
Behind region, the DNA fragmentation of variation is imported with by the insertions such as chemical synthesis only target site.
It can for example be carried out in addition, the site-specific mutation carried out using PCR is imported by double-primer method, described pair is drawn
Thing method refers to:To encode the Double stranded plasmids of part as template, 2 kinds of synthesis oligomerizations containing the variation complementary with+chain and-chain are used
Primer enters performing PCR.
In addition, the DNA of part of the coding comprising multiple domains can be by will it is expected the list of the coding present invention of number
The strand of dna connection of body part (1 domain) is connected and made.For example, just coding includes the DNA of the part of multiple domains company
For connecing method, appropriate restriction enzyme sites can will be imported in DNA sequence dna and be passed through with the double-stranded DNA of limitation enzymatic fragmentation
DNA ligase is attached.Restriction enzyme sites can be a kind, can also import multiple different types of restriction enzyme sites.Or
Person, the DNA of part of the coding comprising multiple domains can also pass through DNA to encoding proteins A (such as International Publication No. 06/
No. 004067) made using above-mentioned variation introductory technique.Here, in the DNA for encoding the part comprising multiple domains, coding
When the base sequence of each monomer part is homologous, homologous recombination may be triggered in host, therefore the coded cell part connected
DNA base sequence between sequence homology be less than 90%, more preferably less than 85%.
Containing the base sequence for encoding aforementioned ligand or the part comprising multiple domains and with energy in the carrier of the present invention
Enough act on the promoter to play a role in host that the mode of the base sequence connects.Generally can be by by afore-mentioned code
The DNA of part is connectable or inertable into carrier and obtained.
, can be with as long as the carrier that the carrier for inserting gene is capable of autonomous replication in host is not particularly limited
Carrier is used as using DNA, phage DNA.On the carrier for inserting gene, such as Escherichia coli are being used as place
When main, pQE systems carrier (QIAGEN company systems), pET systems carrier (Merck company systems) and pGEX systems carrier (GE can be enumerated
Healthcare Japan systems) carrier etc..When using Brevibacillus bacterium as host, such as conduct can be used
PUB110 known to hay bacillus carrier or pHY500 (Japanese Unexamined Patent Publication 2-31682 publications), pNY700 (Japanese Unexamined Patent Publication 4-
No. 278091 publications), pNU211R2L5 (Japanese Unexamined Patent Publication 7-170984 publications), pHT210 (Japanese Unexamined Patent Publication 6-133782
Number publication) or as Escherichia coli and pNCMO2 (the Japanese Unexamined Patent Publication 2002-238569 of the shuttle vector of Brevibacillus bacterium
Number publication) etc..
Host is converted by using carrier, transformant can be obtained.As host, it is not particularly limited, inexpensively a large amount of
In terms of production, Escherichia coli, hay bacillus, Brevibacillus, staphylococcus, streptococcus, streptomycete can be suitably used
Belong to the bacteriums (eubacteria) such as (Streptomyces), corynebacterium (Corynebacterium).Can more preferably withered grass bar
Bacterium, bacillus brevis, staphylococcus, streptococcus, streptomyces (Streptomyces), corynebacterium
(Corynebacterium) gram-positive bacteria such as.Can be further preferably as the suitable examples for being adapted to a large amount of production albumin As
(International Publication No. 06/004067) and known Brevibacillus bacterium.
As Brevibacillus bacterium, it is not particularly limited, such as soil bacillus brevis can be enumerated
(Brevibacillus agri), Potsdam bacillus brevis (Brevibacillus borstelensis), short and small short gemma
Bacillus (Brevibacillus brevis), middle spore bacillus brevis (Brevibacillus centrosporus), bridge stone are short
Bacillus (Brevibacillus choshinensis), beautiful bacillus brevis (Brevibacillus formosus),
Pollute bacillus brevis (Brevibacillus invocatus), side spore bacillus brevis (Brevibacillus
Laterosporus), marsh bacillus brevis (Brevibacillus limnophilus), secondary short and small bacillus brevis
(Brevibacillus parabrevis), Luo Yishi bacillus brevis (Brevibacillus reuszeri), thermophilic red
Bacillus brevis (Brevibacillus thermoruber).It is preferred that short and small bacillus brevis 47 plants (JCM6285), short and small short
Bacillus 47K strains (FERM BP-2308), short and small bacillus brevis 47-5Q strains (JCM8970), bridge stone bacillus brevis
HPD31 strains (FERM BP-1087) and bridge stone bacillus brevis HPD31-OK strains (FERM BP-4573).According to raising output
The purpose of, the Deficient In Extracellular Proteases strain, high expression strain or sporulation capability defect of above-mentioned Brevibacillus bacterium can be used
The variant (or induction strain) of strain etc.If specifically enumerated, can use as the egg from bridge stone bacillus brevis HPD31
The bridge stone bacillus brevis HPD31-OK (Japanese Unexamined Patent Publication 6-296485 publications) of white enzyme variant, without sporulation energy
The bridge stone bacillus brevis HPD31-SP3 (International Publication No. 05/045005) of power.
As the method that carrier is imported to host cell, can enumerate for example:Using the method for calcium ion, electroporation,
Protoplasm body, lithium acetate method, agroinfection, particle bombardment or polyethylene glycol method etc., but it is not limited to these.In addition, make
For the method for the function of the gene obtained by showing host, can also enumerate makes the gene integration that is obtained in the present invention to base
Method because of group (chromosome) etc..
By above-mentioned transformant or DNA cell-free protein synthesis system has been used, part can be manufactured.
When using transformant manufacture part, can by using medium culture transformed cells and in thalline is cultivated (
Including in thalline pericentral siphon region) or nutrient solution in generated (outside thalline), accumulate part and manufacture, can also be adopted from the culture
Collect desired part.
When using transformed cells manufacture part, additionally it is possible to accumulate and match somebody with somebody in the intracellular and/or pericentral siphon region of transformant
Body.In this case, accumulation in the cell when, the oxidation of marking protein can prevented, be also not present and medium component
Side reaction in terms of be favourable, when being accumulated in pericentral siphon region, the decomposition side caused by intracellular protease can suppressed
Face is favourable.On the other hand, when manufacturing part, additionally it is possible to part is secreted into the extracellular of transformant.In this case,
Bacterial cell disruption, the process of extraction are not needed, therefore is favourable in terms of manufacturing cost is reduced.
Carried out with the method for the transformed cells of the medium culture present invention according to the method for being generally used for cultivating host.With
As long as the culture medium of the transformant obtained by culture can high efficiency, produce the part to high receipts amount and be then not particularly limited.
Specifically, glucose, sucrose, glycerine, polyprotein peptone, meat medicinal extract, yeast extract, caseinhydrolysate amino can be used
The carbon sources such as acid, nitrogen source.In addition, the inorganic salts such as sylvite, sodium salt, phosphate, magnesium salts, manganese salt, zinc salt, molysite can be added as needed on
Class.When using the host cell of auxotropic, the required nutriment of addition fertility.In addition, if necessary to
The antibiotic such as penicillin, erythromycin, chloramphenicol, neomycin can be added.
And then decomposed to suppress to be present in the part caused by the protease from host inside and outside thalline, low molecule
Change, can be added with debita spissitudo known to various protease inhibitors, i.e. phenylmethylsulfonyl fluoride (PMSF), benzenecarboximidamide, 4- (2-
Amino-ethyl)-benzene sulfonyl fluorine (AEBSF), antiprotease, chymostatin (Chymostatin), bright Aprotinin,
Pepsin inhibitor A, phosphoramidon, aprotinin (Aprotinin), ethylenediamine tetra-acetic acid (EDTA) and/or other cities
The protease inhibitors sold.
And then in order that part correctly folds, such as can also utilize GroEL/ES, Hsp70/DnaK, Hsp90,
Hsp104/ClpB equimolecular companions.In this case, for example, can be made by coexpression or the gimmick such as fusion protein its with
Body coexists.It should be noted that in order that part correctly folds, also there are as below methods:It will be helpful to the additive correctly folded
It is added in culture medium and carries out the gimmick such as cultivating at low temperature, but is not limited to these.
As culture medium of the culture using Escherichia coli as transformed cells obtained from host, LB culture medium (eggs can be enumerated
White peptone 1%, yeast extract 0.5%, NaCl 1%) or 2 × YT culture mediums (peptone 1.6%, yeast extract 1.0%,
NaCl 0.5%) etc..
As culture medium of the culture using Brevibacillus bacterium as transformant obtained from host, TM cultures can be enumerated
Base (peptone 1%, meat medicinal extract 0.5%, yeast extract 0.2%, glucose 1%, pH7.0) or 2SL culture medium (peptones
4%th, yeast extract 0.5%, glucose 2%, pH7.2) etc..
In addition, by 15~42 DEG C of cultivation temperature, preferably 20~37 DEG C under the conditions of air agitation aerobic culture number
Hour~a couple of days, match somebody with somebody so as to accumulate and reclaim in culture intracellular (including in pericentral siphon region) or culture solution (extracellular)
Body.Ventilation can also according to circumstances be closed and carry out Anaerobic culturel.
, can be after culture terminates by the conventional separation methods such as centrifuging, filtering when secretion produces recombinant protein
The supernatant for cultivating protein of the cell with being produced containing secretion is separated, so as to reclaim produced recombinant protein.
In addition, in culture intracellular (including in pericentral siphon region) accumulation, such as can also be by being utilized from nutrient solution
The methods of centrifuging, filtering is gathered thalline and then is broken the thalline using ultrasonic fragmentation, French cell press method etc.
Broken and/or addition surfactant etc. and it is solubilized, so as to reclaim the part for accumulating production in the cell.
When making part by cell-free protein synthesis system controlling, as cell-free protein synthesis system, do not have
It is particularly limited to, such as synthesis system from prokaryotic, from plant cell, from higher animal cells etc. can be used.
The purifying of part can be separately through affinity chromatography, cation or anion-exchange chromatography, gel filtration chromatography etc.
It is or they are appropriately combined and carry out.
Obtained purifying substance is the confirmation of target ligand this point, can pass through conventional method, such as SDS polyacrylamides
Amine gel electrophoresis, N-terminal amino acid sequence analysis, Western blotting etc. are carried out.
The part that the present invention is used is immobilized onto the carrier formed by water-insoluble base material as affinity ligand, so as to
To manufacture affine isolation medium.Here, " affinity ligand " refers to:Based on being combined into the specific of representative with antigen and antibody
Intermolecular affinity, from a certain elements collection selectively trap (with reference to) target molecule material (functional group) term;This
Refer to the protein combined with specific for immunoglobulin in invention.When " part " is simply expressed as in this specification, also with " parent
And part " equivalent in meaning.
As the carrier formed by water-insoluble base material, can enumerate:The inorganic carriers such as bead, silica gel;It is poly- by being crosslinked
Vinyl alcohol, crosslinked polyacrylate, cross-linked polyacrylamide, crosslinked polystyrene etc. synthesize macromolecule or cellulose, agarose,
The organic carrier that the polysaccharides such as cross-link dextran are formed;And organic-organic, organic and inorganic for obtaining these combinations etc. is multiple
Close carrier etc..As cellulose, crystallinity cellulose, cross-linked cellulose can be enumerated.As agarose, crosslinking fine jade can be enumerated
Lipolysaccharide.
As commercially available product, the GCL2000 as Porous cellulose gel, pi-allyl glucan and methylene can be exemplified
Sephacryl S-1000 that bisacrylamide covalent cross-linking forms, the Toyopearl as methacrylate ester carrier, make
The agarose CL4B of carrier is crosslinked for agarose system and as cellulose-based Cellufine for being crosslinked carrier etc..It is but of the invention
In water insoluble carrier be not limited only to illustrate these carriers.
In addition for water insoluble carrier, from expectation surface area from the point of view of the application target of this affine isolation medium and method
Greatly, it is however preferred to have the Porous of many appropriately sized pores.Can be pearl, monoblock shape, fiber as the form of carrier
Any of shape, membranaceous (including doughnut) etc., can select any form.
On the process for fixation of part, for example, can by using be present in the amino of part, carboxyl or sulfydryl with
Past coupling method is incorporated into carrier.As coupling method, can enumerate:Make carrier and cyanogen bromide, epichlorohydrin, diglycidyl ether,
Toluene sulfochloride, trifluoro ethyl sulfonic chloride, hydrazine and sodium metaperiodate etc. react and by it is support-activated (or to carrier surface import reactivity
Functional group), with as being fixed of part compound carry out coupling reaction so that immobilization method;And to carrier
With the compound as being fixed of part existing for carbodiimide etc condensation reagent or glutaraldehyde are added in system
There is the reagent of multiple functional groups in the molecule, by being condensed, being crosslinked the method and being fixed.
Furthermore, it is possible to the spacer molecule by multiple atomic buildings is imported between part and carrier, can also be on carrier
Direct fixed ligand.Therefore, for immobilization, part can be chemically modified, can also added useful to immobilization
Amino acid residue.As the amino acid useful to immobilization, can enumerate has with the chemical reaction to being immobilized onto side chain
The amino acid of functional group, it can enumerate for example:Side chain contains the Lys of amino, side chain contains the Cys of sulfydryl.As long as to
The effect that body assigns is similarly to assigning in the matrix for forming ligand immobilisation in water insoluble carrier, then to for fixing
The modification of change, remodeling method do not limit.
As the antibody-like protein matter to be purified in the present invention, immunoglobulin G can be enumerated or immunoglobulin G derives
Thing, but it is not limited to these.
As immunoglobulin G, can enumerate:Human IgG1, IgG2, IgG4;Mouse IgG 1, IgG2A, IgG2B, IgG3;
Rat IgG1, IgG2C;Goat IgG 1, IgG2;Cavy IgG;Ox IgG2;Rabbit IgG., can as immunoglobulin G derivative
To enumerate for example:The partial domain of immunoglobulin G is replaced into the domain of the immunoglobulin G of other species and melted
Mosaic type immunoglobulin G after conjunction;By CDR (the Complementarity Determinig of immunoglobulin G
Regions, complementary determining region) aliquot replacement for other species CDR part merge after human-likeization immunoglobulin
G;Immunoglobulin G obtained by molecular modification is subject to the sugar chain in Fc areas;Make the Fv areas of immunoglobulin G and Fc areas are merged and
Artificial immunoglobulin G obtained etc..
In addition, although the region for broadly being combined part is defined as Fab areas (particularly Fv areas) and Fc areas, antibody
Stereochemical structure it is clear and definite, therefore the protein of the object combined as part and affine isolation medium can also be passed through
Protein engineering means Fab areas, Fc areas are implemented on the basis of the stereochemical structure in region for keeping albumin A to be combined into
The protein of one step transformation (fragmentation etc.).
By making antibody-like protein matter be contacted with the affine isolation medium containing the part for being immobilized onto carrier, so that anti-
The process that body sample protein is adsorbed in affine isolation medium;And, connect more than pH3.5 eluent and affine isolation medium
The process touch, eluted antibody-like protein matter, so as to be purified to antibody sample protein.
For the first process of the purification process of antibody-like protein matter, by making antibody-like protein matter and containing immobilization
In the affine isolation medium contact of the part of carrier, so that antibody-like protein matter is adsorbed in affine isolation medium.Specifically,
After the buffer solution containing antibody-like protein matter is adjusted into neutrality, the solution is set to pass through filled with the affine of affine isolation medium
Post, so as to adsorb antibody-like protein matter.As buffer solution, can enumerate for example:Citric acid, 2- (N- morpholinoes) ethyl sulfonic acid
(MES), double (2- ethoxys) amino (trihydroxy methyl) methane (Bis-Tris), N- (2- acetylaminos) imido oxalic acid
(ADA), piperazine -1,4- double (2-ethanesulfonic acid) (PIPES), N- (2- acetylaminos)-Tau (ACES), 3- (N-
Quinoline generation) -2- hydroxy-propanesulfonic acids (MOPSO), N, N- double (2- ethoxys)-Tau (BES), 3- (N- morpholinoes) third sulphurs
Sour (MOPS), (methylol) the methyl-2-amino ethyl sulfonic acids of N- tri- (TES), 4- (2- ethoxys) -1- piperazine ethanesulfonic acids (HEPES),
Triethanolamine, 3- [4- (2- ethoxys) -1- piperazinyls] propane sulfonic acid (EPPS), three (methylol) methylglycines (Tricine),
Trishydroxymethylaminomethane (Tris), glycylglycine, N- bicine N-s, N- tri- (methylol) methyl -3- amino
The buffer solution such as propane sulfonic acid (TAPS) or Dulbecco phosphate buffer normal saline.Antibody-like protein matter is set to be adsorbed in affine separation
PH during matrix is preferably 6.5~8.5, more preferably pH7~8.When the antibody-like protein matter is adsorbed in affine isolation medium
Temperature is preferably 1~40 DEG C, more preferably 4~30 DEG C.
After the first process, appropriate pure buffer solution can be made by affinity column and inside column scrubber.At this
Carve, desired antibody-like protein matter is adsorbed in the affine isolation medium in post.In order to which the buffer solution for washing and using can use
With the buffer solution identical buffer solution used in the first process.
For the second process of the purification process of antibody-like protein matter, more than pH3.5 eluent is set to be separated with affine
Matrix is contacted, and antibody-like protein matter is eluted.As eluent, can enumerate containing such as acetate ion, citrate from
The moon such as son, glycine, succinate ion, phosphate anion, formate ion, propionate ion, γ-aminobutyric acid, lactic acid
The eluent of ionic species.
The pH of eluent is preferably more than pH3.5, more preferably more than pH3.6, more preferably more than pH3.75, is entered
One step is more preferably more than pH3.8, particularly preferably more than pH3.9, most preferably more than pH4.0.The pH of the eluent upper limit
Preferably pH6.0.
When from affine isolation medium antibody elution sample protein, multiple pH eluent can also be used to wash by stages
De- antibody-like protein matter.In addition, if pH is made with gradient elution using different pH two or more eluent (such as pH6 and pH3)
Eluted, then can more highly be purified with gradient, therefore be preferable.The affine isolation medium of the present invention is special
Be not can antibody elution at a high ph, therefore in gradient elution preferably in its partial routine the eluent containing pH4~6.
During absorption, washing when, elution when buffer solution can also add surfactant (such as Tween20, Triton-X100), from
Liquor (such as urea, guanidine), amino acid (such as arginine).
Equally, the pH in the affinity column filled with affine isolation medium during antibody elution sample protein is preferably pH3.5
More than, more preferably more than pH3.6, more preferably more than pH3.75, it is even more preferably more than pH3.8, it is especially excellent
Elect more than pH3.9, most preferably more than pH4.0 as.When being eluted with more than pH3.5 condition, can reduce to antibody
Damage (Ghose S. etc., Biotechnology and bioengineering, 2005, volume 92, No. 6).In addition, washing
The upper limit of the pH in the affinity column filled with affine isolation medium during de- antibody-like protein matter is preferably pH6.0.By this hair
Bright purification process, antibody-like protein matter can be made to be dissociated under the conditions of the acidic elution of more neutral side, washed in acid condition
Eluting peak, elution curve during de- antibody-like protein matter is more sharp.By making eluting peak, the elution curve of chromatogram sharpened, from
And less eluent of the eluent recovery containing high concentration antibody of volume can be used.
Temperature during antibody elution sample protein is preferably 1~40 DEG C, more preferably 4~30 DEG C.
The rate of recovery of the antibody-like protein matter reclaimed by the purification process of the present invention is preferably more than 90%, more preferably
More than 95%.The rate of recovery is calculated by following formula.
The rate of recovery (%)=(concentration (mg/mL) of the antibody-like protein matter eluted × liquid measure (ml) eluted) ÷
(liquid measure (ml) of the concentration (mg/mL) of the antibody-like protein matter of institute's sample introduction × institute's sample introduction) × 100
The purification process of the present invention can reduce being mixed into for the protein from the host for expressing antibody-like protein matter.
Further, it is also possible to reduce being mixed into for the aggregation of antibody-like protein matter.Being mixed into for these protein may make antibody-like protein matter
The burden increase (process number increase, yield reduce) of purification procedures in manufacture, foreign protein may cause serious as medicine
Side effect, the problem can be then avoided using the purification process of the invention of high pH eluent.
When antibody-like protein matter before purification is the mixture with the protein from host cell, of the invention is affine
Isolation medium is for being effective by antibody-like protein matter and Separation of Proteins from host.As host cell proteins matter
The host cell in source is the cell that can express antibody-like protein matter, can particularly enumerate and establish gene recombination technology
Chinese hamster ovary celI, Escherichia coli are as an example.Protein from these hosts can be determined by commercially available immunity detection reagent
Amount.Such as the protein from Chinese hamster ovary celI can be determined using CHO HCP ELISA kits (Cygnus company systems)
Amount.
When antibody-like protein matter before purification is the mixture with the aggregation of antibody-like protein matter, of the invention is affine
Isolation medium from the solution of the aggregation containing antibody-like protein matter, for example relative to the total of the antibody-like protein matter in eluent
The antibody-like protein matter that the solution purification at least containing 1% or 5%, 10% aggregation goes out non-agglomerated is measured, is assembled for removing
It is effective for body.The content of aggregation can for example be analyzed by gel filtration chromatography, be quantitative.
Affine isolation medium can by make not damage completely ligand compound, carrier base material function degree it is suitable
When highly acid or strong basicity pure buffer solution (being also the solution containing appropriate denaturant or organic solvent sometimes) from
Wherein by being washed, so as to recycling.
Part and affine isolation medium can for example be swashed to the compatibility of antibody sample protein by using surface plasma
The biology sensors such as the Biacore systems (GE Healthcare Japan systems) of first resonance principle are tested.On part institute
Compatibility having, to immunoglobulin, by Biacore system measurements described later to immunoglobulin G preparation
During compatibility, binding constant (KA) it is preferably 106(M-1) more than, more preferably 107(M-1) more than.
As condition determination, as long as the bar of binding signal of the ligand binding when Fc areas of immunoglobulin can be detected
Part, can be by being measured simply to enter under the neutrallty condition of temperature 20~40 DEG C (steady temperatures), pH6~8
Row evaluation.
As the immunoglobulin molecules for combining object, can enumerate for example:Gamma Globulin as polyclonal antibody
Monoclonal antibody in " Nichiyaku " (immunoglobulin G) (Japanese pharmaceutical companies' system), street drug.
On affinity difference, those skilled in the art can easily test in such a way:With identical
Condition determination obtains the association reaction curve to identical immunoglobulin molecules, using the incorporating parametric obtained when parsing compared with
Object part is compared, so as to test.
As incorporating parametric, such as binding constant (K can be usedA), dissociation constant (KD) (work such as field, " raw body Wu Quality forever
The リ ア Le タ イ system Xie Xi experiment method that interacts (the real time parsing experimental method of biological substance interaction) ", Springer-
Verlag Tokyo, 1998, page 41).Part and Fab affinity costant can be obtained as follows using Biacore systems:Passing
Immobilization belongs to the Fab fragments of the immunoglobulin of VH3 subfamilies on sensor chip, is used under conditions of 25 DEG C of temperature, pH7.4
The experimental system added with part is obtained in stream.It should be noted that binding constant is expressed as sometimes in some documents
Affinity costant, both definition are essentially identical.
Embodiment
The present invention is described in more detail below based on embodiment, but the present invention is not limited by these embodiments.The present embodiment
In, the making of recombinant DNA, operation etc. are then implemented as long as no special declaration according to following experimental programs.(1)
T.Maniatis, E.F.Fritsch, J.Sambrook work, " Molecular Cloning/A Laboratory Manual ",
Second edition (1989), Cold Spring Harbor Laboratory periodicals (U.S.).(2) village pine is positive writes " lab guide in fact
Genetic engineering ", the 3rd edition (1996), Wan Shan Co., Ltd. publish.In addition, the reagent used in the present embodiment, restriction enzyme etc. are only
Commercially available product is then used without special declaration.
For the various protein obtained in embodiment, with " variation (the wild type for the letter of statement domain-imported
Be then Wild) " form state.For example, the wild type C-structure domain of albumin A is expressed as " C-wild ", variation G29E is imported with
C-structure domain variant be expressed as " C-G29E ".On being imported with the statement of 2 kinds of variants to make a variation simultaneously, then with oblique line simultaneously
Described in row.For example, the C-structure domain variant for being imported with variation G29E and the S13L that makes a variation is expressed as " C-G29E/S13L ".In addition,
The protein that multiple single domains are formed by connecting then adds connected number after single domain (period) and " d " carrys out table
State.For example, the protein that 5 C-structure domain variants for being imported with the variation G29E and S13L that makes a variation are formed by connecting is expressed as " C-
G29E/S13L.5d”。
(embodiment 1) evaluates the antibody binding capacity of C-structure domain variant using IgG fixation supports
Using way of bailment, fully synthetic reworked C-G29A.2d synthetic gene (Eurofins Genomics
K.K. make), the gene is imported with the C-G29A.2d's (sequence number 6) of G29A variations in C-structure domain body of the coding to albumin A
It is imported with the DNA (sequence number 7) that DNA 5 ' ends addition PstI recognition sites, 3 ' ends addition XbaI recognition sites forms
Amino acid replacement variation described in table 1.Expression plasmid restriction enzyme PstI and XbaI (precious biotech firms after this is subcloned
System) digestion, the DNA fragmentation of acquirement is connected to the bacillus brevis expression carrier pNCMO2 with identical limitation enzymic digestion
In (precious biotech firm's system), so as to prepare in bacillus brevis expression carrier pNCMO2 inserted with coding reworked C-
The DNA of G29A.2d amino acid sequence expression plasmid.It should be noted that Escherichia coli are used in the preparation of plasmid
JM109 strains.
With resulting plasmid conversion bridge stone bacillus brevis SP3 strains (precious biotech firm's system), secretion manufacturing improvement is cultivated
Type C-G29A.2d genetic recombinants.The genetic recombinants are (more with the 30mL of the neomycin containing 60 μ g/mL A culture mediums
Polyprotein peptone 3.0%, yeast extract 0.5%, glucose 3%, magnesium sulfate 0.01%, ferric sulfate 0.001%, manganese chloride
0.001%th, zinc chloride 0.0001%) in 30 DEG C of shaken cultivations 3 days.
C-Q9A/G29A.2d, C-Q9S/G29A.2d, C-Q9T/G29A.2d, the C-G29A/ that will be expressed by above-mentioned steps
Q32A.2d, C-G29A/K35S.2d, C-G29A/Q32T.2d amino acid sequence be recorded in respectively the sequence number 10 of sequence table~
15。
After culture, by centrifuging (15000rpm, 25 DEG C, 5 minutes) after nutrient solution removes thalline, efficient liquid phase is used
The concentration of reworked C-G29A.2d in chromatographic determination culture supernatant.Implement to cultivate according to following conditions with IgG fixation supports
The elution test of reworked C-G29A.2d and C-G29A.2d in supernatant.
< uses the condition > of the elution test of IgG fixation supports
Carrier:IgG Sehparose FF (GE Healthcare company systems)
Post:OMNIFIT posts (Diba Industries company systems), column diameter 0.66cm, post bed height 6.4cm, cylinder
Product:2.19mL
Flow velocity:0.8mL/ minutes, time of contact 2.7 minutes
Applied sample amount:470 μ L (ligand concentration 1.3mg/mL)
Equilibrating buffer solution:50mM Tris hydrochloric acid 150mM NaCl pH of buffer 7.5
Elution requirement:50mM citrate buffer solution pH6.0 → 50mM citrate buffer solutions pH3.0 (20CV)
Calculate the elution pH of reworked C-G29A.2d when on the basis of C-G29A.2d elution pH difference.Result is shown
In table 1.Any reworked C-G29A.2d is high from the pH of IgG fixation supports elution compared with C-G29A.2d.The result table
It is bright:The carrier that immobilization has reworked C-G29A.2d can elute anti-under the higher pH of the carrier than being fixed with C-G29A.2d
Body.
[table 1]
(embodiment 2) evaluates the antibody binding capacity of C-structure domain variant using intermolecular interaction resolver
Use (the GE Healthcare public affairs of biology sensor Biacore 3000 that make use of surface plasmon resonance
Department's system) various protein and immunoglobulin acquired in parsing embodiment 1 compatibility.In the present embodiment, using by people
The immunoglobulin G preparation (being hereafter denoted as human IgG) that plasma fraction goes out.
Human IgG is immobilized onto sensor chip, various protein is flowed on chip, detects both interactions.
Immobilization of the human IgG to sensor chip CM5 is by using n-hydroxysuccinimide (NHS) and N- ethyls-N '-(3- bis-
Methylaminopropyl) the amine coupling method of carbodiimide hydrochloride (EDC) carries out, closed using monoethanolamine (sensor chip, solid
Determining is GE Healthcare company systems with reagent).Human IgG solution is by gamma Globulin " Nichiyaku " (Japanese pharmaceutical
Company system) according to 1.0mg/mL it is dissolved in standard buffer solution (20mM NaH2PO4-Na2HPO4, 150mM NaCl, pH7.4) and make
Standby.By human IgG solution immobilization buffer solution (10mM CH3COOH-CH3COONa, pH5.0) 100 times of dilution, according to
Human IgG is fixed on sensor chip by specification subsidiary Biacore 3000.In addition, for another flowing on chip
Pond, the processing of monoethanolamine is fixed after being activated by EDC/NHS, so as to prepare the reference pond as negative control.] various
Protein running buffer (20mM NaH2PO4-Na2HPO4, 150mM NaCl, 0.005%P-20, pH7.4) appropriate prepare
Scope (for various protein, preparing the different solution of 3 kinds of protein concentrations) into 10~1000nM, by each protein solution
Added 30 seconds to sensor chip with the μ L/ minutes of flow velocity 20.At 25 DEG C of measurement temperature successively observation addition when (with reference to phase, 30
Second) and addition terminate after (dissociate phase, 60 seconds) association reaction curve.Each observation adds Glycine-HCl10mM after terminating
PH3.0 (GE Healthcare company systems) makes sensor chip regeneration (30 seconds), and (purpose is that removing residues in sensor chip
On addition protein, confirm that the binding activity of the human IgG of institute immobilization is substantially completely recovered).
For resulting association reaction curve (subtracting the association reaction curve after the association reaction curve with reference to pond), make
Carried out being based on 1 with the subsidiary software BIA evaluation of system:The fitting parsing of 1 binding model, it is normal to calculate association rate
Number (kon), dissociation rate constant (koff) and binding constant (KA=kon/koff).Show the result in table 2.
As shown in table 2, reworked C-G29A.2d is equal journey with the incorporating parametric of human IgG with C-G29A.2d (control)
Degree.Specifically, the binding constant with human IgG is illustrated as 10 in the case of any part8M-1More than.Any reworked C-
G29A.2d is respectively provided with the antibody binding capacity with importing the C-G29A.2d equal extents before making a variation in neutral pH range.
[table 2]
(embodiment 3) evaluates the antibody binding capacity of B structure domain variant using IgG fixation supports
Using way of bailment, fully synthetic reworked B-Q9A/G29A.2d synthetic gene (Eurofins
Genomics K.K. systems), the gene is imported with the B-G29A.2d (sequences of G29A variations in coding to the B structure domain body of albumin A
Number 8) in the DNA (sequence number 9) that DNA 5 ' ends insertion PstI recognition sites, 3 ' ends insertion XbaI recognition sites forms,
The Gln of 9 is replaced as Ala.Recombinate, express similarly to Example 1, for obtained culture supernatant, carried with IgG immobilizations
Body implements elution test.As a result, the pH that B-Q9A/G29A.2d compared with B-G29A.2d, elutes from IgG fixation supports
Improve 0.22.The result shows:The variation of embodiment 1 is not limited only to C-structure domain, and same effect is also generated to B structure domain
Fruit.
(embodiment 4)
The culture supernatant of C-G29A.2d for the reworked C-G29A.2d prepared in embodiment 1 and as control, makes
Implement elution test according to following conditions with IgG fixation supports.
< uses the condition > of the elution test of IgG fixation supports
Carrier:IgG Sehparose FF (GE Healthcare company systems),
Post:OMNIFIT posts (Diba Industries systems), column diameter 0.66cm, post height of bed 6.4cm
Column volume:2.19mL、
Flow velocity:0.8mL/ minutes, time of contact 2.7 minutes
Applied sample amount:470 μ L (ligand concentration 1.3mg/mL)
Equilibrating buffer solution:50mM Tris hydrochloric acid 150mM NaCl pH of buffer 7.5
Elution requirement:Elute (1) 50mM citrate buffer solutions pH4.0 (2CV), elution (2) 50mM citrate buffer solutions
pH3.0(4CV)
Determine the ligand concentration of each eluent and calculate the rate of recovery.Show the result in table 3.Any reworked C-G29A.2d
Compared with C-G29A.2d, the rate of recovery of the eluent under pH4.0 is high.It can be predicted by the result:There is C- with immobilization
G29A.2d carrier is compared, and the antibody recovery rate when carrier that immobilization has reworked C-G29A.2d elutes under higher pH carries
It is high.
[table 3]
The antibody elution experiment of (embodiment 5) reworked C-G29A.2d is affine isolation medium
The reworked C-G29A.2d and the C-G29A.2d of control culture cultivated similarly to Example 1 is centrifuged
Separation, isolates thalline, acetic acid is added into obtained culture supernatant after pH is adjusted into 4.5, standing one hour makes target
Protein precipitation.By centrifuging recovery precipitation, buffer solution (50mM Tris-HCl, pH8.5) is dissolved in.
Then, by using the anion exchange color of HiTrap Q posts (GE Healthcare Bio-Sciences systems)
Compose protein of interest matter.Specifically, target protein solution is made an addition to anion exchange buffer A (50mM
Tris-HCl, pH8.0) HiTrap Q posts after equilibrating, after being washed with anion exchange with buffer A, point take by using
The salt concentration gradient of anion exchange buffer A and anion exchange buffer B (50mM Tris-HCl, 1M NaCl, pH8.0)
The target protein eluted in the process.The target protein solution taken will be divided to dialyse ultra-pure water, after dialysis
The aqueous solution as final purification of samples.It should be noted that the protein purification carried out by using the chromatogram of post is utilized
AKTA avant systems (GE Healthcare Bio-Sciences systems) are implemented.
As water-insoluble base material, the commercially available pre- shape post of activated form " Hitrap NHS activated HP " 1mL are used
(GE Healthcare company systems).The post is using Sepharose as matrix and is imported with the N- of proteinacious ligand immobilisation
HOSu NHS (NHS) base.It is affine so as to prepare respectively according to the final purification of samples of product handbook immobilization as part
Isolation medium.
Specifically, prepare 1mL by final purification of samples coupling buffer (0.2M sodium carbonate, 0.5M NaCl,
PH8.3) it is diluted to final concentration about 13mg/mL solution.1mM HCl after being cooled with an ice bath are shunted with flow velocity 1mL/ minutes
2mL, the operation is carried out 3 times and removes the isopropanol in post.The sample prepared before this with identical flow velocity addition immediately after is dilute
Solution 1mL is released, by the closing up and down of post, 30 minutes are stood at 25 DEG C, so that by acquired protein immobilization in post.Then
Closing is released, coupling buffer 3mL is flowed into identical flow velocity, reclaims unreacted protein.Then, closing buffer solution will be flowed into
(0.5M monoethanolamines, 0.5M NaCl, pH8.3) 2mL operation is implemented 3 times, will flow into washing buffer solution (0.1M acetic acid, 0.5M
NaCl, pH4.0) 2mL operation implemented 3 times, finally flow into standard buffer solution (20mM NaH2PO4-Na2HPO4、150mM
NaCl, pH7.4) 2mL, so as to complete the making of affine splitter.Using resulting affine isolation medium, according to following conditions
Implement antibody elution experiment.As control, the affine isolation mediums of C-G29A.2d equally prepared are also tested.Measure is washed
The absorbance of de- liquid simultaneously calculates antibody recovery rate.
The condition > that < is tested using the antibody elution of the affine isolation mediums of reworked C-G29A.2d
Post:Pre- shape post Hitrap NHS activated HP " 1mL (GE Healthcare company systems) (include immobilization
There is the post of the carrier of each part)
Flow velocity:0.33mL/ minutes, 3.0 minutes times of contact,
Sample solution:Gamma Globulin " Nichiyaku " (Japanese pharmaceutical companies' system) 5mL (ligand concentration 1mg/mL)
Equilibrating buffer solution:Dulbecco ' s phosphate buffer normal salines (Sigma-Aldrich company systems)
Elution requirement:Elute (1):50mM citrate buffer solutions (4CV), experiment A:PH4.0, experiment B:PH3.75, experiment
C:PH3.5, elution (2):50mM citrate buffer solutions pH3.0 (4CV)
Measurement result is shown in table 4.Compared with C-G29A.2d affine isolation medium, prepared using C-Q9T/G29A.2d
Affine isolation medium under high pH (pH4.0~3.5) antibody recovery rate in eluent it is high.The result shows:In embodiment 4
The experiment using IgG agaroses in the high part of the rate of recovery under pH4.0, be made being immobilized onto water insoluble carrier
During affine isolation medium, it is possible to increase the antibody recovery rate under high pH elution requirement.
[table 4]
Sequence table
<110>Kanegafuchi Chemical Ind (KANEKA CORPORATION)
<120>The purification process of antibody-like protein matter
<130> B150472WO01
<150> JP 2015-145007
<151> 2015-07-22
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 56
<212> PRT
<213>Staphylococcus aureus (Staphylococcus aureus)
<400> 1
Ala Gln His Asp Glu Ala Gln Gln Asn Ala Phe Tyr Gln Val Leu Asn
1 5 10 15
Met Pro Asn Leu Asn Ala Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu
20 25 30
Lys Asp Asp Pro Ser Gln Ser Ala Asn Val Leu Gly Glu Ala Gln Lys
35 40 45
Leu Asn Asp Ser Gln Ala Pro Lys
50 55
<210> 2
<211> 61
<212> PRT
<213>Staphylococcus aureus (Staphylococcus aureus)
<400> 2
Ala Asp Ala Gln Gln Asn Lys Phe Asn Lys Asp Gln Gln Ser Ala Phe
1 5 10 15
Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly
20 25 30
Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu
35 40 45
Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys
50 55 60
<210> 3
<211> 58
<212> PRT
<213>Staphylococcus aureus (Staphylococcus aureus)
<400> 3
Ala Asp Asn Asn Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile
1 5 10 15
Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln
20 25 30
Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala
35 40 45
Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys
50 55
<210> 4
<211> 58
<212> PRT
<213>Staphylococcus aureus (Staphylococcus aureus)
<400> 4
Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile
1 5 10 15
Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln
20 25 30
Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala
35 40 45
Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys
50 55
<210> 5
<211> 58
<212> PRT
<213>Staphylococcus aureus (Staphylococcus aureus)
<400> 5
Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile
1 5 10 15
Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Gly Phe Ile Gln
20 25 30
Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala
35 40 45
Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys
50 55
<210> 6
<211> 116
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223>C domains are mutated
<400> 6
Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile
1 5 10 15
Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln
20 25 30
Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala
35 40 45
Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ala Asp Asn Lys Phe Asn
50 55 60
Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu
65 70 75 80
Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro
85 90 95
Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala
100 105 110
Gln Ala Pro Lys
115
<210> 7
<211> 359
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<221> mutation
<223>C domains are mutated
<400> 7
ctgcagataa caaatttaac aaagaacaac aaaacgcttt ctacgaaatc ctgcacttgc 60
caaaccttac tgaagaacaa cgtaatgctt tcatccaatc cctgaaagat gatccatctg 120
tatccaaaga aattttggca gaggctaaaa aacttaacga cgctcaggcg cctaaggctg 180
ataacaaatt caataaagaa cagcaaaacg ctttttatga aatccttcac ctgccaaatc 240
ttacagaaga acaacgcaac gcattcattc aaagcttgaa ggatgaccct tccgttagca 300
aagagatcct ggctgaagca aaaaagttga atgatgcgca agcaccaaaa taatctaga 359
<210> 8
<211> 116
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223>B domains are mutated
<400> 8
Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile
1 5 10 15
Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln
20 25 30
Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala
35 40 45
Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ala Asp Asn Lys Phe Asn
50 55 60
Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu
65 70 75 80
Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro
85 90 95
Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala
100 105 110
Gln Ala Pro Lys
115
<210> 9
<211> 359
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<221> mutation
<223>B domains are mutated
<400> 9
ctgcagataa caaatttaac aaagaacaac aaaacgcttt ctacgaaatc ctgcacttgc 60
caaaccttaa tgaagaacaa cgtaatgctt tcatccaatc cctgaaagat gatccatctc 120
aatccgctaa ccttttggca gaggctaaaa aacttaacga cgctcaggcg cctaaggctg 180
ataacaaatt caataaagaa cagcaaaacg ctttttatga aatccttcac ctgccaaatc 240
ttaacgaaga acaacgcaac gcattcattc aaagcttgaa ggatgaccct tcccaaagcg 300
caaatttgct ggctgaagca aaaaagttga atgatgcgca agcaccaaaa taatctaga 359
<210> 10
<211> 116
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> C-Q9A/G29A.2d
<400> 10
Ala Asp Asn Lys Phe Asn Lys Glu Ala Gln Asn Ala Phe Tyr Glu Ile
1 5 10 15
Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln
20 25 30
Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala
35 40 45
Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ala Asp Asn Lys Phe Asn
50 55 60
Lys Glu Ala Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu
65 70 75 80
Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro
85 90 95
Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala
100 105 110
Gln Ala Pro Lys
115
<210> 11
<211> 116
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> C-Q9S/G29A.2d
<400> 11
Ala Asp Asn Lys Phe Asn Lys Glu Ser Gln Asn Ala Phe Tyr Glu Ile
1 5 10 15
Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln
20 25 30
Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala
35 40 45
Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ala Asp Asn Lys Phe Asn
50 55 60
Lys Glu Ser Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu
65 70 75 80
Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro
85 90 95
Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala
100 105 110
Gln Ala Pro Lys
115
<210> 12
<211> 116
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> C-Q9T/G29A.2d
<400> 12
Ala Asp Asn Lys Phe Asn Lys Glu Thr Gln Asn Ala Phe Tyr Glu Ile
1 5 10 15
Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln
20 25 30
Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala
35 40 45
Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ala Asp Asn Lys Phe Asn
50 55 60
Lys Glu Thr Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu
65 70 75 80
Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro
85 90 95
Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala
100 105 110
Gln Ala Pro Lys
115
<210> 13
<211> 116
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> C-G29A/Q32A.2d
<400> 13
Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile
1 5 10 15
Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Ala
20 25 30
Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala
35 40 45
Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ala Asp Asn Lys Phe Asn
50 55 60
Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu
65 70 75 80
Thr Glu Glu Gln Arg Asn Ala Phe Ile Ala Ser Leu Lys Asp Asp Pro
85 90 95
Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala
100 105 110
Gln Ala Pro Lys
115
<210> 14
<211> 116
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> C-G29A/K35S.2d
<400> 14
Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile
1 5 10 15
Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln
20 25 30
Ser Leu Ser Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala
35 40 45
Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ala Asp Asn Lys Phe Asn
50 55 60
Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu
65 70 75 80
Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Ser Asp Asp Pro
85 90 95
Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala
100 105 110
Gln Ala Pro Lys
115
<210> 15
<211> 116
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> C-G29A/Q32T.2d
<400> 15
Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile
1 5 10 15
Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Thr
20 25 30
Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala
35 40 45
Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ala Asp Asn Lys Phe Asn
50 55 60
Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu
65 70 75 80
Thr Glu Glu Gln Arg Asn Ala Phe Ile Thr Ser Leu Lys Asp Asp Pro
85 90 95
Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala
100 105 110
Gln Ala Pro Lys
115
Claims (10)
1. a kind of purification process of antibody-like protein matter, it includes following (a)~(b) process:
(a) antibody-like protein matter is made to be contacted with the affine isolation medium containing the part for being immobilized onto carrier, so that antibody sample
The process that protein is adsorbed in affine isolation medium;With
(b) more than pH3.5 eluent is made to be contacted with affine isolation medium, the process that antibody-like protein matter is eluted;
The part contains will in E, D, A, B or the amino acid sequence in C-structure domain described in sequence number 1~5 from albumin A
The Gln and/or Lys of Fc binding sites are replaced into amino acid sequence obtained from Ala, Ser and/or Thr, and
Compared with the part before the displacement, the antibody binding capacity in acid pH range reduces the part.
2. purification process according to claim 1, wherein,
The affine isolation medium is the carrier for having in water-insoluble base material immobilization part.
3. purification process according to claim 1 or 2, wherein,
Water-insoluble base material contains synthesis macromolecule or polysaccharide.
4. purification process according to claim 3, wherein,
Polysaccharide is cellulose or agarose.
5. according to purification process according to any one of claims 1 to 4, wherein,
Eluent is containing selected from by acetate ion, citrate ion, glycine, succinate ion, phosphate anion
With the acidic buffer of at least one kind of anionic species in the group of formate ion composition.
6. according to purification process according to any one of claims 1 to 5, wherein,
The content of the aggregation of the antibody-like protein matter of protein from host and/or immunoglobulin in eluent subtracts
It is few.
7. purification process according to claim 6, wherein,
The elution of antibody-like protein matter is eluted using pH gradient and carried out.
8. purification process according to claim 7, wherein,
PH gradient elutes to be carried out using the eluent of pH4~6.
9. according to purification process according to any one of claims 1 to 8, wherein,
Antibody-like protein matter before purification is the mixture with the protein from host cell.
10. according to purification process according to any one of claims 1 to 8, wherein,
Antibody-like protein matter before purification is the mixture with the aggregation of antibody-like protein matter.
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CN112334490A (en) * | 2018-06-20 | 2021-02-05 | Jsr株式会社 | Affinity vectors using variant VHH antibodies |
CN112969708A (en) * | 2018-11-12 | 2021-06-15 | 纳维格蛋白质有限公司 | Novel triple-helical polypeptides lacking immunoglobulin FC domain binding affinity and uses thereof |
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GB202113626D0 (en) | 2021-09-24 | 2021-11-10 | Cytiva Bioprocess R & D Ab | FC binding polypeptides |
CN114409765A (en) * | 2021-12-23 | 2022-04-29 | 苏州创胜医药集团有限公司 | Method for purifying antibody |
GB202203478D0 (en) | 2022-03-14 | 2022-04-27 | Cytiva Bioprocess R & D Ab | Vh3 binding polypeptides |
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Also Published As
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US20180215785A1 (en) | 2018-08-02 |
JPWO2017014261A1 (en) | 2018-04-26 |
WO2017014261A1 (en) | 2017-01-26 |
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